Cryopreservation of avian semen is a useful tool to preserve genetic resource for aim of preventing extinction induced by infectious disease like avian influenza. Unlike those of mammals, data from chicken cryopreserved semen has not been showed feasible results. So, various cryoprotectants and diluents have been examined in many methods. In this report, as a major ingredient of avian seminal plasm, glutamine was substituted by alanyl glutamine to enhance physiological stability of chicken semen during freezing. We studied effect of glycerol and Dimethylacetamide(DMA) on motility and progressive motility of spermatozoa using glutamine diluent(EK-G) or alanyl glutamine diluent(EK-A) condition. The semen of Ogye was collected twice a week by the dorso-abdominimal massage method and diluted with same volume of EK-G or EK-A at 25℃ and stored for 10 min at 4℃ in cold chamber. Glycerol or DMA was added to diluted semen to reached 7% of final concentration at 4℃. After 3min of equilibration, the diluted semen was packed into 0.25ml straws and subjected to cryopreservation used freezing equipment. The packed straw were placed on height 5 cm above surface of liquid nitrogen(LN2) and held for 10min. After preserved for 2 weeks, the straw was thawed onto the 4℃ cooling bath. The images of motility and progressive motility spermatozoa were recorded by digital image recorder and analyzed by manual. The results showed 68.5% motility and 34.1% progressive motility in DMA/EKA diluent, 31.45% and 17.6% in glycerol/EKA, 45.4% and 8.6% in DMA/EKG, and 9.7% and 6.4% in glycerol/EKG. With these results, the alanyl glutamine and DMA could be used as a main composition of diluent and cryoprotectant for cryopreservation of chicken semen.

The aim of this study was to examine the effect of acteoside (the cyclin-dependent kinase inhibitor) on the SCNT efficiency with adult fibroblasts in dog. Canine adult fibroblasts were obtained from muscle and cell cycle of fibroblasts was synchronized by culturing to confluency, serum starvation and treating with 30 μM acteoside for 48 h. Cell cycle stages, cell cytotoxicity (apoptosis) and, prduction of reactive oxygen species (ROS) were analyzed using flow cytometry. The canine cells, prepared by confluent-cell culture or treating with 30 μM acteoside for 48 h, were injected into enucleated in vivo matured oocytes, the couplets were electrical fused and activated by calcium ionomycin. SCNT embryos using acteoside-treated fibroblasts were surgically transferred into oviducts of estrus cycle synchronized recipient dogs. In cell cycle synchronization (G0/G1), there was no significant difference between serum starvations (83.9%) and acteoside treated groups (81.3%) that were higher than confluent group (78.5%). In production of apoptosis, confluent and acteoside treated groups (4.3 and 4.5%, respectively) were generated less than serum starvation group (21.8%). In case of ROS, serum starvation group was induced a significantly higher than other groups. After synchronization of the donor cell cycle, either confluent or acteoside treated, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into naturally estrus-synchronized surrogates. Fusion and cleavage rate of acteoside treated group were 64.1 and 41.5%, which were higher than those of confluent group (53.9 and 20.6%, respectively). The reconstructed embryo development rates to 4-cell and 8-cell in acteoside treated group were 29.5 and 14.8%, respectively, while confluent group showed 11.1 and 3.2%, respectively. Total 54 SCNT embryos using acteoside-treated fibroblasts were transferred into oviducts of 2 recipient dogs and one recipient finally delivered one puppy, whereas din`t detected pregnancy on transfer of cloned embryos reconstructed with confluent cells in 6 surrogate dogs. In conclusion, the results of the current study demonstrated that canine fibroblasts could be successfully arrested at the G0/G1 stage with reduced the formation of ROS and apoptosis after acteoside treatment. This results may contribute to improve the effi-ciency of canine SCNT. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Acteoside acts as an anti-oxidative activity and anti-apoptosis in the cells. But, it has been not studied on maturation and development of porcine oocytes. The aims of the present study were to examine the effects of acteoside on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. Oocytes were matured in tissue culture medium-199, supplemented with acteoside at various concentrations: 0 (control), 10, 30 and 50 μM. The oocytes maturation rates of groups supplemented with acteoside were no significantly different (81.13, 85.96, 82.95 and 83.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (44.83 vs. 27.75%). And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. In the results. during IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with control oocytes. And reverse transcription polymerase chain reaction (RTPCR) of parthenogenetic blstocysts revealed that acteoside increased the anti-apoptotic genes (Mcl-1, Bcl-2 and Bcl-xL), whereas reduced the expression of pro-apoptotic genes (Bax and Bak). In conclusion, based on the results, the effect of acteoside on IVM was not attractive. However, in acteoside treated group, cytoplasmic maturation seemed to be improved with morphologically uniform distribution of cytoplasmic organelles. Furthermore, embryonic development in acteoside treated group was significantly highly increased than that of non-treated group. Our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes, providing a improved method for porcine oocytes in vitro. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

The objective of the current study was to describe in vitro embryo production in Hanwoo, analyzing oocytes yield and embryo production. The effects of oocytes production and the number of OPU procedures per animal on embryo production were also evaluated. OPU was done every 3～4 days during experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, we compared the recovery rate of oocytes based on OPU session (Experiment 1). The average of collected oocytes was calculated from every 10 session. The average number of total oocytes recovered per animalonsessionwas 5.16 (mean). Second, we compared the recovery rate base on collection period of OPU (Experiment 2). The following results show the difference of the number of recovered oocytes in every month during the procedure between the months of session. Every animal shows the constant number of recovered oocytes for the first 5 months. However, the recovery rate of oocytes was decreased from month 6 to 8. Third, we compared the developmental rate to blastocyst in two groups (Experiment 3). Oocytes by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24～48 h after in vitro fertilization (IVF) was 75.8% and blastocyst development rate was 18.8% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (61.1%), blastocyst development rate was higher compared with needle puncture group (28.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio- technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being.

Cytokines are known to function as regulatory molecules that can be produced by virtually every nucleated cell type in the body, including lymphocytes, monocytes/macrophages, epithelial cells, fibroblasts, and many others. Cytokines include lymphocyte-derived factors (lymphokines), monocyte-derived factors (monokines), hematopoietic factors (colony-stimulating factors), connective tissue/ growth factors, and chemotactic chemokines. Cytokines released in response to infection can affect tumor development in different ways. When exposed to infectious agents, cytokines are secreted by sentinel cells, such as macrophages and dendritic cells. These cytokines include interleukin 1 (IL-1) and tumor necrosis factor-α, as well as others, such as IL-6, IL-12, and IL-18. When released in sufficient quantities, these molecules can cause inflammation. Chronic inflammation is highly associated with tumor initiation, promotion, and progression. In this article, we review the roles and mechanisms of cytokines in tumor development.

Gene targeting is a genetic technique that utilizes homologous recombination between an engineered exogenous DNA fragments with the endogenous genome of an organism. In domestic animal, gene targeting has provided an important tool for producing Knock-out pig for GGTA1 gene to use xenotransplantation. The frequency of homologous recombination is a critical parameter for the success of gene targeting. The efficiency of homologous recombination in somatic cells is lower than that in mouse ES cells. So the application of gene targeting in somatic cells has been limited by its low efficiency. Recently, knock-out rat and mouse was generated by introducing nonhomologous end joining (NHE)-mediated deletion or insertion at the target site using zinc-finger nucleases (ZFN). Therefore, the development of effective knock-out and knock-in techniques in domestic animal is very important in biomedical research. In this present study, we investigated whether homologous recombination events occurs at cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene locus using ZFN in porcine primary fibroblast. CMAH-targeted ZFN DNA and mRNA were purchased from SIGMA-Aldrich. CMAH neo targeting vector consists of the neomycin resistance gene as a positive selectable marker gene, 789 bp 5’ arm and 763 bp 3’ arm from Exon 8 of CMAH gene. For transfection, the targeting vector and ZFN DNA or mRNA were introduced into ear fibroblasts cells of Chicago miniature pig by electroporation. After selection of G-418, PCR analysis was performed using 213 colonies transfected with ZFN DNA or mRNA. As a result, 39 positive colonies were identified in colonies transfected with ZFN DNA or mRNA. To our knowledge, this study provides the first evidence that the efficiency of gene targeting using ZFN was higher than that of conventional gene targeting in the porcine fibroblast. These cell lines may be used in the production of CMAH knock-out for xenotransplantation.

The objective of this study was to evaluate in vitro production of bovine embryos in Hanwoo. Oocytes were collected by ovum pick up (OPU) from ovaries of genetically high-value Hanwoo or by needle puncture from ovaries of slaughtered cattle. OPU was done every 3 4 days duing experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, We compared the in vitro maturation rate in two groups (Experiment 1). 545 oocytes were recoverd from 4 females by 32 trials of OPU and then 433 oocytes were shown MⅡ stage after in vitro maturation (79.4%). In case of needle puncture group, 1905 oocytes were collected and then 1420 oocytes were matured to MⅡ stage during in vitro culture(74.5%). Second, we compared the developmental rate to blastocyst in two groups (Experiment 2). 1420 oocyte by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24 48 h after in vitro fertilization (IVF) was 88.6% and blastocyst development rate was 20.5% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (84.8%), blastocyst development rate was higher compared with needle puncture group (26.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio-technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being. This research was suppoted by Imsil-gun agricultural technology service center.

The use of natural paint for the application to walls and furnishings is now increasing to improve indoor air quality, thereby the natural paint could be a significant source of biogenic volatile organic compounds (BVOCs) in indoor environments. Recent studies have shown that gas-phase reactions between terpenes and ozone can generate sub-micron size particles and toxic volatile organic compounds such as aldehydes and ketones. In this research, we have studied the formation of particles and secondary organic compounds during the reaction of ozone with terpenes emitted from commercial natural paint. The paint applied onto stainless steel was dried and oxidized in a teflon chamber. Two monoterpenes (α- and β-pinenes) were identified by FTIR and GC/MS. Several tests were performed to evaluate the effects of ozone concentration on particle formation. Increased ozone levels significantly affect the increase of particle number concentration (monitored with SMPS), which results in the increase of particle counts ranging from 8,000 to 70,000 particles/㎤. Gas-phase products such as formaldehyde, acetaldehyde, acetone + acrolein, and propionaldehyde were identified during the terpene/ozone reactions by HPLC. These compounds are potential hazardous chemical compounds having harmful health effects to animals and plants. The results obtained from this study provide an insight on the adverse effect of eco-friendly natural product on indoor air quality (IAQ).

Background : The purpose of this study was to evaluate methods to high quality food ramie rice cake, thereby increasing farm income. This study investigated the effects of different Split Application and Adapted Fertilizer Culture for Stable Production with Edible Leaf in Ramie (Boehmeria nivea L.). Methods and Results : Method of fertilizer application were conducted under three condition compose to standard application (N-P-K-Compost applied at 27-9-27-1,500 ㎏․ 10 a−1), 25% increased application (N-P-K-Compost applied at 34-11-34-1,500 ㎏․10 a−1), 50% increased application (N-P-K-Compost applied at 41-14-41-1,500 ㎏․ 10 a−1) cultivation. Compost and fused phosphate applied at 100% of basal fertilizer in March 25. The split application level of standard application (N-K) applied at 50% of basal fertilizer in March 25, 50% of top dressing were five times application (N-K) in May 4, July 5, September 5, October 5, 20% respectively. Transplanting with Yeongkwang varieties in April 12 by growing pot seedling for 35 days in 2011. Planting density were spaced 60 ㎝ apart in rows 25 ㎝ apart with rice straw cutting covered open cultivation. Plot design randomized block 3 repetition. Conclusion : Organic matter, available phosphate, K2O, CaO, and MgO of soil increased in all plots with the addition of fertilizers culture, specifically in 25% and 50% increased application. Growth characteristics by 50% of top dressing were five times application (N-K) in May 4, July 5, September 5, October 5, compared to standard application (N-K) applied at 100% basal fertilizer, aerial part as a result were so many such amount of growth as number of branch and leaf per plant, etc., Length, width of leaf and leaf colorimetry of aboveground part growth increment rising highest. Green leaf yields compared to standard fertilizer 100% application (1,344 kg․ 10 a−1). 25% and 50% increased application (N-K) applied at 50% basal fertilizer, 50% of top dressing were five times application increased by 9%, 17%. respectively.

Background : Angelica dahurica Bentham et Hooker, Angelica gigas Nakai, Ostericum koreanum Maximowicz and Peucedanum japonicum Thumberg　are a major medicinal plant in north Geungbuk province. Using medicinal plants are impotant it`s ingredient. Dry condition and stroage method are not standard manual. The ingredient variation of dry condition and stroage method were not researched. Methods and Results : Using plant material were cutivated on Gyongsangbukdo Bonghwa area. It were studied ingredient variation after dry and storage condition by HPLC methods. Major ingredient of Angelica gigas Nakai are decurusin, decurusinangelate. Heated air bulk dry get more decursin than natuarl dry and decurusinangelate of natural bulk dry was higher than heated air bulk dry. Major ingredient of Ostericum koreanum Maximowicz are imperatorin and isoimperatorin.. Imperatorin of Ostericum koreanum was highest peak on 50℃ heated-air dry after plastic bag sorage and isoimperatorin was highest peak on 40℃ heated-air dry after mountain cultivation. Imperatorin is a major ingredient Angelica dahurica Bentham et Hooker. Heated air bulk dry get more decursin and decursinangelate than natuarl dry and small heated-air dry. Peucedanol-7o_glucoside is a major ingredient Peucedanum japonicum Thumberg. Natural bulk dry get more peucedanol-7o_glucoside than heated-air bulk dry. Conclusion : Ingredient of Angelica dahurica Bentham et Hooker, Angelica gigas Nakai, Ostericum koreanum Maximowicz are different under various cutivation, drying method, storage. Diffent Ingedients of Angelica gigas Nakai, Ostericum koreanum Maximowicz were not accord it’s optical conditon.

Background : Curcuma longa L., in the family Zingiberaceae, is distributed in tropical and/or sub-tropical regions mainly in India and China. This species is commonly called turmeric, powder is used as medicinal herbs and/or flavor enhancer. It has been cultivated in southern region mainly Jindo. However, it might be possible to extend cultivation regions due to rise in average temperature. In order to select superior lines based on agronomic characteristics, we analyzed multivariate and estimated selection effects from C. longa germplasm. Methods and Results : The C. longa germplasm were cultivated in an experimental field located in Eumseong, NIHHS, RDA. The harvested roots were investigated in agronomic characteristics included in yield and then considered its relationship among the 9 germplasm by multivariate analysis method. Results from principal component analysis (PCoA) showed that it represented 70.00% and 80.44% accumulated explanation from four and five principal compounds (PC). PCoA was conducted from 9 agronomic characteristics and then correlation coefficient has been showed by analysis between each main component value and agronomic characteristics. Value of the first PC was 2.25, 24.96% explanation of total dispersion, plant height, number of rootlet and weight of rootlet were correlated with a somewhat higher level as 0.41, 0.43 and 0.52. Value of the fifth PC was 0.94, 10.43% explanation of total dispersion, the number of shoots was correlated with a higher level as 0.87. Selection effects with outstanding candidate lines including higher lines were estimated at 126.13% in yield. Conclusion : These data on multivariate based on agronomic characteristics will be give us invaluable breeding information by selection of superior lines.

Background : Water uptake and flow across cellular membranes is a fundamental requirement for plant growth and development, and plant water status is important not only for plant growth under favorable conditions but also for ability of a plant to tolerate adverse environmental conditions. Thus identification of plasma membrane water channel genes (aquaporins) in ginseng provides extensive information for functional studies and the development of markers for salinity stress tolerance. Methods and Results : For salinity treatment, the plants were grown for 4 weeks in culture medium gelled with 0.8% Phytoagar, and the old media were replaced with the fresh medium containing NaCl at 0, 50, 100, 200 and 400 mM, respectively. The samples for stress treated and non-stressed plants were collected from 6h to 72h, and frozen immediately into liquid nitrogen. According to the sequence information from the assembled transcripts, four primer pairs were designed from the aquaporin gene regions. In order to determine the pattern of aquaporins expression in ginseng seedlings to salinity stress, we conducted semi-quantitative RT-PCR. Conclusion : A tonoplast intrinsic protein 1 (TIP1)-type aquaporin is not only believed to be essential for plant life, but also to be beneficial for growth under salinity stress. Therefore, a deeper understanding of aquaporin genes in ginseng will be essential for crop improvement, which could help us to understand the molecular genetic basis for the ginseng genetic improvement and also provide the functional genetic resources for selective breeding and transgenic research.

Background : This study was carried out to understand the effect of seedling weight (SW) on growth and flowering in Panax ginseng. Methods and Results : The testing materials were Chunpoong (CP), Yunpoong (YP) and Jakyeongjong (JK). The increase of seedling (1yr) weight led to an increase in ratio of flowering plant and in number of flower per plant. The seed setting rate of two year-old plant (CP, YP, JK) increased with increase of SW at the planting time (PT) and number of flower per plant of three year-old plant (CP, YP) increased also. In the two year-old plant (JK), the ratio of three leaves per plant was 8.8, 19.6, 31.0, 42.0, 44.7 and 58.2%, respectively, in the SW of >0.6, 0.6~0.8, 0.8~1.0, 1.02~1.2, 1.2~1.4 and 1.4g<. The growth of ginseng plant was good with increase of SW at the PT. Conclusion : There was a highly positive correlation between seedling weight and flowering characteristics.

Background : An important feature of the nutrient solution is that they affect not only the growth but also quality of crops by changing nutrient uptake, especially due to changes of EC in nutrient solution. This study was carried out to investigate effect of EC in nutrient solution on growth and ginsenoside of ginseng. Methods and Results : EC in nutrient solution was controlled with 0.68, 0.84, 1.23, 1.41 dS/m. The root weight of ginseng treated by low EC levels in nutrient solution was higher during the initial of growth. However, the higher EC levels, the more increased the change rate of root weight from the initial to the middle of growth. The highest amount of ginsenoside was changed by growth period. Although the total amount of ginsenoside in root is highest treated by EC 0.68 dS/m at 45 days after treatment. the total amount of ginsenoside in root is highest treated by EC 1.23 dS/m at 135 days after treatment. Conclusions : EC in nutrient solution should to be controlled depending on the stage of growth and the part of use, i.e. root and leaves, when ginseng is cultivated through nutri-culture.

Background : Boron (B) is an essential element required for the growth of plant. It has a narrow range of optimal concentration from minimum to maximum thresholds than other micro-elements. The study was carried out to investigate to the influence of B excess concentrations on physiological disorder of leaf, growth and mineral concentration of ginseng to obtain basic information for physiological disorder diagnose. Methods and Results : The ginseng cultivar ‘Gumpoong’ was cultivated by hydroponic system for 2 months. The toxicity symptoms which ginseng leaves were curved downwardly and induced to chlorosis after beginning to dry the edge appeared on leaves more than 30 ppm compared to the control(3 ppm). The growth of ginseng was more decreased with higher B concentration. Mn uptake was also decreased as B concentration increased. It was found that B excess hindered the growth of ginseng and was reversely related to Mn uptake. Conclusions : More than B 30 ppm can negatively affect growth and mineral uptake. Consequently, B excess can occur physiological disorder of ginseng.

Background : Cylindrocarpon root rot, caused by Cylindrocarpon destructans and Fusarium solani, is a major disease which lead to replanting failure in ginseng garden. Chlamydospores of C. destructans, generated by poor environmental condition, can survived more than ten years in soil without host plant, ginseng. Density of soil pathogens gradually decreased as the progress of time since ginseng was harvested. Methods and Results : Soil chemical properties was analyzed from soil samples of 43 regions in farmer’s ginseng garden to study the variation of content and the correlation among inorganic contents. Soil samples of 24 regions was also analyzed to study correlation between progressed-year after ginseng harvest and soil chemical properties. Variation of soil chemical properties in descending order was Fe, Zn, P2O4, NO3, Mn. The content of Fe and Zn were great variation among inorganic chemicals of soil of farmer’s field. Electrical conductivity to induce physiological demage in excessive concentration showed highly significant positive correlation with the content of NO3 and K. As the progress of year after ginseng harvest, the content of organic matter and zinc was increased, while pH and Na were decreased in farmer’s field to cultivate ginseng. There were highly significant positive correlation between the progress of year after ginseng harvest and zinc content in farmer’s field to cultivate ginseng. Ratio of root rot of 2-year-old ginseng showed significant positive correlation with K content, and negative correlation in experimental field cultured by six rotation crops for one year. Conclusion : Root rot by soil pathogens was closely related with the content of potassium and zinc in soil chemical properties.

Background : Root diseases caused by Cylindrocarpon destructans and Fusarium solani decrease the yield and quality of ginseng. Cylindrocarpon root rot is a major disease caused by replanting failure in ginseng garden. Methods and Results : Solarization was done in the infested soil of the greenhouse for summer season (from July 24 to Autumn 31, 2014) after putting green manure (Sudan grass) and calcium cyanamide (CC) into the soil. Mycelium and conidia of C. destructans died at 4 0℃ after 15 hours, and 45℃ after 5 h, but it did not die at 35℃ after 15 h. Those of C. destructans died after keeping it for 2 hours daily at 40℃ for 9 days, and 45℃ for 8 days, but did not die at 38℃ for 9 days. Maximum soil temperature was 55.4℃ in 5 cm depth, 48.7℃ in 10 cm, 44.7℃ in 15 cm, 42.5℃ in 20 cm, and 31.9℃ in 30 cm by putting green manure into the soil and solarization. Reduction of sudan grass increased electrical conductivity (EC), organic matter, P2O5, K, and Mg, while decreased pH, NO3-N, and Na. Addition of calcium cyanamide and urea gave a negative effect on the growth of ginseng because EC and NO3-N were increased excessively than the optimal range. Solarization using green manure mixed with CC was the most effective in decreasing soil-borne pathogens of 2-year-old ginseng. But the root disease that occurred between single treatment of sudan grass and the treatment mixed with calcium cyanamide showed not a significant different. Addition of calcium cyanamide showed the decrease of root weight because leaves were dead early by a excessive increase of EC and NO3-N. Conclusion: Soil disinfection using green manure and solarization in greenhouse was effective in inhibiting root rot, however, it did not completely kill the soil-borne pathogens.